The invention is related to a method for investigating the imperishability of liquid foodstuffs by means of electron spin resonance (ESR), wherein a sample of the foodstuff is exposed to an elevated temperature, as compared to room temperature, for an extended period of time, wherein during the period of time an ESR signal of the sample is measured in predetermined time intervals, the intensity of the ESR signal is plotted vs. time and the moment in time is detected when the intensity shows a superproportional increase.
The invention is, further, related to an apparatus for investigating the imperishability of liquid foodstuffs by means of electron spin resonance (ESR), wherein a sample of the foodstuff is exposed to an elevated temperature, as compared to room temperature, for an extended period of time, wherein during the period of time an ESR signal of the sample is measured in predetermined time intervals, the intensity of the ESR signal is plotted vs. time and the moment in time is detected when the intensity shows a superproportional increase.
When doing so, the samples are provided with a so-called spin-trap-substance or a spin-label-substance or a corresponding intrinsic substance is already comprised in the sample anyhow.
A method of the afore-mentioned kind is disclosed in an article by Kaneda, H. et al. xe2x80x9cFree Radical Reactions in Beer during Pasteurizationxe2x80x9d, International Journal of Food Science and Technology (1994), 29, pp. 195 to 200. A corresponding method as well as an apparatus for executing same is also disclosed in EP 0 720 026 A2. Still another description of such a method together with a corresponding apparatus may be found in an application note by Barr, D. xe2x80x9cMeasuring Flavor Stability of Beer using the Bruker EMX Spectrometerxe2x80x9d, Bruker EPR Application Note (1998).
For a better understanding of the present method the underlying measuring technology of electron spin resonance shall be briefly discussed.
Electron spin resonance (ESR) is a partial field within the general field of magnetic resonance. In ESR, a sample substance is simultaneously exposed to a high frequency electromagnetic field as well as to a strong constant magnetic field of high homogeneity. By varying the field strength of the constant magnetic field various electron spin resonances are excited within the sample and may be plotted as a spectrum. Typically a high frequency electromagnetic field within X-band, i.e. at about 10 GHz is utilized, which means that the strength of the constant magnetic field is about 0.32 T. It is, however, also known to conduct ESR measurements on the one hand at frequencies being ten times as high or, on the other hand, being only one tenth of the afore-mentioned frequeny.
As mentioned before, for exciting ESR it is only necessary to expose the sample substance to a magnetic high frequency field. When the measuring frequencies are within the microwave range, hollow cavities are conventionally utilized for that purpose. Such cavities are mostly rectangular cavities of the oscillation mode TE10n, however, in some instances also cylindrical cavities of the oscillation mode TE011 are utilized.
Such rectangular cavities are conventionally configured in double length, i.e. as TE102 cavities. The sample container having the shape of a sample tube is then inserted at the cavity center where the H-lines of both oscillation loops extend parallel to each other. In such a situation the sample, is, hence, located within the area of maximum magnetic high frequency field strength. Due to the characteristics of such oscillation mode, this is also the area of minimum electric high frequency field strength. This is of particular advantage for lossy samples because in such a way the dielectric losses are at a minimum.
When liquid samples are to be investigated with ESR measurements, one has to bear in mind that such samples per se are subject to substantial dielectric losses. One has, therefore, to take care that the liquid sample substance is as much as possible arranged within the plane within which the magnetic high frequency field strength is at a maximum and the electric high frequency field strength is at a minimum. For achieving that one may either utilize one single thin capillary, however, it is also well known to utilize so-called xe2x80x9cflat cellsxe2x80x9d, i.e. sample tubes which have been flattened within the measuring area. By doing so the liquid sample substance is essentially distributed within an area so that one may place relatively much sample substance within the afore-mentioned plane of maximum magnetic and minimum electric high frequency field strength.
Sample heads of the afore-mentioned kind are generally known and are also commercially available from the competent manufacturers.
It is, furthermore, well known in the field of magnetic resonance to arrange a plurality of different samples within independent vessels or at least at individual locations within the same probe head being distant from each other, for conducting comparative measurements. By doing so one may either conduct direct comparative measurements between two sample substances or one may utilize one of the substances, being a reference substance, as a standard, whereas only the other substance is investigated. This standard may be utilized for quantitative scaling purposes or one may use same for effecting a field control (so-called xe2x80x9cinternal lockxe2x80x9d). Within this context various probe heads have been disclosed, permitting that two distinct sample substances are received within a sample area or within two independent sample areas, respectively.
An example of such a prior art probe head is disclosed in an article by DALAL et al. xe2x80x9cThe effects of lossy solvents on quantitative EPR studiesxe2x80x9d, Journal of Magnetic Resonance, 44, pp. 415-428 (1981).
In this prior art probe head (cf. FIG. 3 in the afore-mentioned article) a sample tube arrangement is utilized in which within a thin-walled NMR-sample tube of 0.5 cm outer diameter a first capillary having an inner radius of 0.05 cm and, either a quartz tube having a radius of 0.064 cm or another capillary having an inner radius of 0.05 cm are utilized. The first capillary contains the sample substance being dissolved within a non-lossy solvent as a reference substance, whereas the second capillary or the tube contains the lossy solvent being conventionally used. Both samples may be arranged within the plane of minimum electric high frequency field strength.
U.S. patent specification No. 5,552,709 discloses a probe head for nuclear magnetic resonance (NMR). This prior art probe head is of elongate design and comprises a plurality of parallel capillaries. The capillary walls consist of a non-conductive material so that the quality factor Q of the resonance system shall be increased by reducing the electrical losses caused by currents flowing radially with respect to the sample container axis.
In another sample container for an NMR-spectrometer, as disclosed in U.S. Pat. specification No. 5,469,061 a plurality of laterally adjacent capillaries is likewise used as a sample container. These capillaries are switched in series such that a flow cell is configured in which the liquid sample substance enters through one capillary, then flows through all of the remaining capillaries under repetitive change of flow direction and, finally, is discharged from another capillary.
Finally, U.S. Pat. specification No. 5,596,276 discloses a standard rectangular cavity as used for electron spin resonance spectrometers.
The afore-mentioned probe heads, therefore, are exclusively intended to be used for making comparative measurements between two samples, one of which being conventionally known for reference purposes.
While ESR measurements on liquid samples have only played a secondary role in the past, because ESR was mostly used as an analytical instrument in connection with solids, various applications have gained attention in the recent past in which ESR was utilized on liquid samples.
One of these applications is the investigation of the imperishability of beer. One has found that the aging of beer, in particular the so-called xe2x80x9cturning sourxe2x80x9d of beer into a condition where it may no more be consumed, is caused by the generation of various aldehydes being side products of reactions in which free radicals are involved. These are processes being comparable to those that also occur in other foodstuffs, for example meat and dairy products.
The behavior of free radicals is in so far highly depending on temperature, wherein processes develop the slower, the lower the storage temperature, for example of beer, is. It is known that beer comprises naturally abundant antioxidants counteracting the afore-mentioned free radicals within such processes. By means of ESR it is possible to investigate the behavior of free radicals, by utilizing the so-called xe2x80x9cspin-trappingxe2x80x9d or the xe2x80x9cspin-labellingxe2x80x9d method. Within this method a beer sample is first de-gassed and, subsequently, a xe2x80x9cspin-trappingxe2x80x9d or a xe2x80x9cspin-labellingxe2x80x9d substance, respectively, is added. The beer sample thus configured is then subjected to a thermal stress program by bringing same to an elevated temperature of e.g. 60xc2x0 C., for thus imitating the aging process in quick motion. By periodically measuring ESR, e.g. every ten minutes, the aging process within the beer may be monitored. By doing so it is possible within a relatively short period of time to find an indication about whether the investigated beer will perish sooner or later. This, again, allows to influence the brewing process by repeatedly measuring in the afore-mentioned way for finally extending the imperishability of the beer. In other sample materials the respective substances are already abundant as intrinsic substances.
The afore-mentioned method is disclosed with further details in the articles of Hirotaka and Barr, mentioned at the outset, as well as in EP 0 720 026 A2 so that reference may be made to these publications insofar.
In order to use ESR measurements on liquid samples, for example in the afore-described application (investigation of imperishability of foodstuffs, or of fermented liquids, especially beer or e.g. wine or dairy products), it is necessary to work with relatively large sample quantities in order to obtain sufficiently high signals as soon as possible. Moreover, the respective method shall allow an automatic execution on a large scale.
With the prior art probe heads for ESR spectrometers allowing measurements on liquid samples, this is not easily possible because these probe heads are normally apparatuses to be used in scientific laboratories, being difficult to handle and requiring a high amount of skill and, in particular, a considerable measuring time.
A significant disadvantage of the prior art method and the corresponding apparatus as described in the three printed publications mentioned at the outset, is that a substantial period of time is necessary for conducting the measurements. It may be that the required amount of time is tolerable for the measurement of one single kind of beer (in order to keep on with this example), the required amount of time is nor more tolerable when a plurality of samples of different foodstuffs (beers) is intended to be measured. This may, e.g. be the case in a large brewery where beers of same or distinct beer kinds being brewed in different tanks shall be monitored continuously during the fermentation process, for if need be, influencing the fermentation process in order to achieve the forecasted imperishability of the beer.
If the prior art method and apparatus were used e.g. for measuring six different samples of distinct foodstuffs (beers) a measuring time corresponding to six times the time of a single measurement would be required. Considering, however, that already a simple measurement requires a period of time of the order of one hour, a measuring result would be available only after several hours. However, then it would already be too late for influencing the production process (fermentation process) of the foodstuff.
Of course, it would be possible in such instances to multiply the number of measuring apparatuses. However, this option may be eliminated due to economical considerations because ESR spectrometers are highly complex, and, hence, very expensive measuring instruments.
It is, therefore, an object underlying the invention to improve a method and an apparatus of the type specified at the outset, such that simultaneous measurements on a plurality of samples becomes possible such that different foodstuffs (beers) may be measured almost parallel in time without the necessity of providing further measuring installations therefore.
In a method of the type specified at the outset, this object is achieved according to the invention in that a predetermined number n of samples of different foodstuffs is investigated simultaneously by executing the following steps within a common sample vessel:
a) filling a first sample of a first foodstuff into the sample vessel;
b) investigating the first sample by means of ESR and measuring the intensity of the ESR signal;
c) plotting the intensity measured in step b) as a first measuring point in a first diagram;
d) discharging the first sample;
e) repeating nxe2x88x921 times steps a) through d) with the nxe2x88x921 samples of the other foodstuffs, wherein in step c) the measured intensities of each foodstuff are plotted into separate diagrams as first points, the number n being dimensioned such that executing steps a) through d) n times corresponds essentially to the predetermined time interval;
f) filling another first sample of the first foodstuff into the sample vessel;
g) investigating the further first sample by means of ESR and measuring the intensity of the ESR signal;
h) plotting the intensity measured in step g) as a second measuring point into the first diagram;
i) discharging the further first sample;
j) repeating nxe2x88x921 times steps e) through i) with the nxe2x88x921 samples of the other foodstuffs, wherein in step h) the measured intensities of each foodstuff are plotted into separate diagrams as second points into the separate diagrams; and
k) repeating steps e) through j) for third and more measuring points until a predetermined number m of measuring points per diagram has been attained.
In an apparatus of the type specified at the outset, the object underlying the invention is achieved in that for simultaneously investigating a predetermined number n of samples of different foodstuffs a common sample vessel is provided and that the apparatus comprises:
a) means for filling a first sample of a first foodstuff into the sample vessel;
b) means for investigating the first sample by means of ESR and measuring the intensity of the ESR signal;
c) means for plotting the intensity measured in step b) as a first measuring point in a first diagram;
d) means for discharging the first sample;
e) means for repeating nxe2x88x921 times steps a) through d) with the nxe2x88x921 samples of the other foodstuffs, wherein in step c) the measured intensities of each foodstuff are plotted into separate diagrams as first points, the number n being dimensioned such that executing steps a) through d) n times corresponds essentially to the predetermined time interval;
f) means for filling another first sample of the first foodstuff into the sample vessel;
g) means for investigating the further first sample by means of ESR and measuring the intensity of the ESR signal;
h) means for plotting the intensity measured in step g) as a second measuring point into the first diagram;
i) means for discharging the further first sample;
j) means for repeating nxe2x88x921 times steps e) through i) with the nxe2x88x921 sample s of the other foodstuffs, wherein in step h) the measured intensities of each foodstuff are plotted into separate diagrams as second points into the separate diagrams; and
k) means for repeating steps e) through j) for third and more measuring points until a predetermined number m of measuring points per diagram has been attained.
It had already been mentioned that a spin-trap substance or a spin-label substance or a corresponding intrinsic substance was added to the sample beforehand.
The object underlying the invention is thus entirely solved.
During the execution of the inventive method and during the use of the inventive apparatus, respectively, a substantial gain in time is obtained, as compared with conventional measurements. The measurements on the individual samples are namely interlocked in time relative to each other with the smallest possible time-offset, such that the entire measuring time is only a little bit longer as the conventional measuring time for one sample. According to the invention, the trick is to utilize the intermission between two individual measurements on the sample in conventional measurements.
Therefore, although only a minimum complication of the apparatus is required, one may measure a plurality of samples of distinct foodstuffs practically parallel in time with one single ESR spectrometer.
According to a first alternative of the invention, the samples are recirculated after discharging so that, in other words, always the same samples are measured within predetermined time intervals.
However, on the other hand it is also possible to dispose of the samples after discharging such that the samples of a specific foodstuff are taken as individual samples from a supply of the specific foodstuff.
Whereas in the first-mentioned case the apparatus has to be configured a little bit more complicated, only a very small amount of sample is required on the other hand because the same sample is repeatedly measured. This is possible because electron spin resonance is a non-destructive measuring technique.
According to the second mentioned alternative, the apparatus may be configured more simple, however, a larger amount of sample material must be at hand, namely a volume of sample material being bigger by a factor corresponding to the number of planet measuring points.
According to another embodiment of the invention the sample vessel is preferably rinsed after each individual measurement.
This measure has the advantage that errors within the measuring values are avoided which may possibly be caused by the fact that after the discharge of a single sample residual amounts of that sample will remain within the sample vessel.
Within the scope of the present invention it is particularly preferred when the samples are measured within a resonator adapted to allow propagation of an electromagnetic field, the sample container being elongate and being located within the resonator along the direction of the magnetic high frequency or radio frequency (RF) field strength at the location of minimum electric RF field strength, that the foodstuffs sample contained in the sample container has minimum dimensions in the direction of increasing electric RF field strength, and that the samples are measured in a sample container having a plurality of elongate sample areas with minimum radial dimensions.
This measure has the advantage that measurements may be conducted with minimum dielectric losses corresponding to a high quality factor of the measuring circuit. This is particularly important in connection with the very narrow-band measuring circuits (resonators) as used in electron spin resonance spectroscopy because already relatively small dielectric losses will result in a dramatic decrease of the quality factor Q. This distinguishes electron spin resonance fundamentally from nuclear resonance utilizing resonators configured as coils having an essentially lower quality factor such that electric losses will influence the quality factor to a much lesser extend.
In further embodiments of the invention the sample areas are arranged parallel and the sample is filled into one end of the sample areas and is discharged from the opposite end of the sample areas.
As an alternative, the sample areas may be arranged in series, the sample is filled into an open end of the first sample area within the series and is discharged from the open end of the last sample area within the series.
Both alternatives have the advantage of a flow cell being particularly adapted for ESR measurements on liquid samples because due to the thin capillaries used only very small dielectric losses occur.
Insofar one has to state that surprisingly the electromagnetic losses being caused by a liquid sample in an ESR probehead do not quite only depend on the amount of sample material and on the positioning of the sample material within the resonator. Instead, one has found that the negative influence of these losses may be drastically reduced by distributing the desired entire sample substance over a plurality of individual amounts of sample and by placing same individually into elongate sample areas within the resonator. Due to the particular field distribution within the resonator and due to the spin effects resulting therefrom and due to the extremely small radial conductive paths within the liquid sample material, the losses are substantially reduced.
The electromagnetic losses being effective in this regard do not only comprise the well-known dielectric losses. In addition, one has to bear in mind that the magnetic component of the electromagnetic high frequency field also generates eddy currents. These shall be avoided or minimized, respectively, as best as one can. In a conductive liquid the magnetic high frequency field namely results in shielding currents about the field direction, namely essentially at the corresponding sample surface. Due to these shielding currents the intended penetration of the magnetic field into the interior of the sample is rendered more difficult which, in turn, results in a signal decrease. If, however, the sample is segmented, e.g. by subdividing same into filaments, the eddy currents are interrupted. This, in turn, results in an increase penetration of the magnetic field into the interior of the sample and, hence, in a signal enhancement.
As a consequence, due to the invention the quality factor of the resonator is much less influenced as before which, finally, has the result that with a relatively large amount of sample substance a corresponding strong measuring signal may be used.
This opens up entirely new fields of applications within industrial production processes because it is no more necessary to prepare a sample with utmost precision. Instead relatively simple apparatuses and less skilled personal are sufficient to conduct quantitatively sufficient measurements.
Therefore, the invention may, further, be used with advantage in a method of the type specified at the outset for investigating liquid samples, when a probe head of the afore-mentioned kind is utilized. As already mentioned before, this holds true, for example, for applications as the investigation of the imperishability of liquid and solid foodstuffs, in particular drinks, preferably beer.
In a preferred embodiment of the inventive probe head the probe head comprises a block being provided with a plurality of axial bores.
This measure has the advantage that a stable and reproducible sample container is available which may be handled, filled and discharged simply.
The block is preferably essentially cylindrical, however, in other embodiments of the invention may also be essentially flat.
As an alternative instead of using a block provided with bores one may also be utilize a bundle of capillaries.
This measure has the advantage that capillaries are industrially available as finished elements so that the mentioned bundles of capillaries may be produced relatively simply and at low cost.
With probe heads of the mentioned type it is particularly preferred when the ratio of the sum of the radial cross-sectional areas of the sample areas and the entire cross-sectional area of the sample vessel is between 0.2 and 0.6.
This ratio has turned out to be optimal both with respect to the maximization of the sample volume and with respect to the stability of the sample vessel.
The sample areas are preferably essentially cylindrical.
This measure has the advantage that the sample vessels may be manufactured relatively easily.
If the probe head is operated at a conventional ESR measuring frequency within the X-band, e.g. at 10 GHz, the diameter of the sample areas is between 0.3 and 1.0 mm, preferably between 0.5 and 0.8 mm.
In that case, it is further, preferred when for an essentially cylindrical sample vessel the outer diameter is between 2.5 and 5 mm preferably between 2.8 and 3.8 mm.
It goes without saying that these dimensions are related to the mentioned frequency band, whereas for lower frequencies (e.g. S-band) or for much higher frequencies (Q-band or V-band) the dimensions must be selected correspondingly larger or smaller, respectively.
For the afore-mentioned configurations it is, further preferred when the cylindrical sample areas within the cylindrical probe head are arranged along the so-called hexagonal densest packing, i.e. when either seven or nineteen sample areas are arranged about a center axis.
In a further embodiment of the invention a pump is connected to the discharge end of the sample areas. This measure has the advantage that the filling and the discharging of the sample areas within the sample vessel may be effected reliably and quickly.
This holds true in particular when the pump is a peristaltic pump.
In further embodiments of the invention it is preferred when the filling-in end of the sample areas is connected to the output of a sampler, and inputs of the sampler are connected to a plurality of containers being filled with different foodstuffs.
This measure has the advantage that the sequential filling of the sample areas of the sample vessel with samples of distinct foodstuffs is effected in an automatized and remote-controlled way.
With this embodiment of the invention one may, finally, provide that a further input of the sampler is connected to a vessel adapted to be filled with a rinsing liquid.
This measure has the advantage that after each measuring process, as mentioned above, a rinsing process may take place for cleaning the sample areas of the sample vessel.
Further advantages will become apparent from the description and the enclosed drawing.
It goes without saying that the features mentioned before and those that will be mentioned here and after may not only be used in the particularly given combination, but also in other combinations or alone without leaving the scope of the present invention.